Although humoral immunity is a critical component in an effective HIV/AIDS vaccine to control the rampant spread of HIV-1, few of the vaccine candidates currently in preclinical and clinical trials appear to elicit broadly neutralizing antibodies. Several lines of evidences show that high mannose type carbohydrates on the HIV-1 glycoprotein gp120 are a novel and promising target to develop broadly neutralizing antibodies. First, high mannose glycans on gp120 play a critical role in HIV transmission and infection through interaction with immune cells, including dendritic cells. Second, one of the few broadly neutralizing HIV monoclonal antibodies, 2G12, recognizes a conformational epitope of high mannose type glycans on gp120. Third, a potent inhibitor of HIV, Cyanovirin-N, binds to terminal a1,2-linked mannose residues on high mannose glycans. Last, the carbohydrates on gp120 are well exposed and conserved among different HIV-1 subtypes and strains. To target the high mannose glycans on gp120, we have created N-glycosylation mutants of S. cerevisiae, identified several yeast glycoproteins that cross-react with 2G12, elicited immune sera that cross-react with Env glycoproteins of HIV-1 and SIV, and neutralized HIV-1 pseudoviruses. The gp120 binding and pseudovirus neutralization activities of these sera are broad, but the antibody titer and neutralization potency of the immune sera need to be improved. We hypothesize that higher levels of Mana1,2Man-specific antibodies with stronger gp120 binding and neutralizing activities can be induced in some of the proposed multiple groups of animals immunized with this new vaccine formulation. The Specific Aims are, (1) optimization of immunization procedures with the whole cells from triple mutant yeast of S. cerevisiae to elicit stronger gp120 cross-reactive and neutralizing antibodies;and (2) analyses of antigenicity and immunogenicity of 2G12 cross-reactive yeast glycoproteins, and elicitation of potent and broadly neutralizing antibodies using a prime-boost strategy. If success, this novel formulation of immunogen from yeast mutant strain will be a safe, cheap, and effective HIV/AIDS vaccine against a broad range of HIV-1 primary isolates. PUBLIC HEALTH RELEVANCE: Although humoral immunity is a critical component in an effective HIV/AIDS vaccine to control the rampant spread of HIV-1, few of the vaccine candidates currently in preclinical and clinical trials appear to elicit broadly neutralizing antibodies. Several lines of evidences show that high mannose type carbohydrates on the HIV-1 glycoprotein gp120 are a novel and promising target to develop broadly neutralizing antibodies. First, high mannose glycans on gp120 play a critical role in HIV transmission and infection through interaction with immune cells, including dendritic cells. Second, one of the few broadly neutralizing HIV monoclonal antibodies, 2G12, recognizes a conformational epitope of high mannose type glycans on gp120. Third, a potent inhibitor of HIV, Cyanovirin-N, binds to terminal a1,2-linked mannose residues on high mannose glycans. Last, the carbohydrates on gp120 are well exposed and conserved among different HIV-1 subtypes and strains. To target the high mannose glycans on gp120, we have created N-glycosylation mutants of S. cerevisiae, identified several yeast glycoproteins that cross-react with 2G12, elicited immune sera that cross-react with Env glycoproteins of HIV-1 and SIV, and neutralized HIV-1 pseudoviruses. The gp120 binding and pseudovirus neutralization activities of these sera are broad, but the antibody titer and neutralization potency of the immune sera need to be improved. We hypothesize that higher levels of Mana 1,2Man-specific antibodies with stronger gp120 binding and neutralizing activities can be induced in some of the proposed multiple groups of animals immunized with this new vaccine formulation. The Specific Aims are, (1) optimization of immunization procedures with the whole cells from triple mutant yeast of S. cerevisiae to elicit stronger gp120 cross-reactive and neutralizing antibodies;and (2) analyses of antigenicity and immunogenicity of 2G12 cross-reactive yeast glycoproteins, and elicitation of potent and broadly neutralizing antibodies using a prime-boost strategy. If success, this novel formulation of immunogen from yeast mutant strain will be a safe, cheap, and effective HIV/AIDS vaccine against a broad range of HIV-1 primary isolates.